Use of a protein of the CRMP family for treating diseases related to the immune system

ABSTRACT

The invention concerns the use of at least a protein of the CRMP family, a polypeptide fragment or an active biological derivative thereof, a nucleotide sequence or sequence fragment coding for said protein, an antisense sequence capable of being specifically hybridized with a sequence with nucleotide-type encoding of said protein or an antibody directed against said protein for producing a medicine for treating diseases related to a dysfunction of the immune system. The invention also concerns a method for diagnosing an autoimmune pathology which consists in measuring the expression of a CRMP protein in the lymphocytes.

The present invention relates to a novel use of so-called ‘CRMPs’(Collapsin Response Mediator Proteins) for the diagnosis and treatmentof pathologies related to a dysfunction of the immune system.

CRMPs, also known as TOAD-64 (Minturn et al, 1995, J Neurosci 15:6757-6766), DRP (dihydropyrimidinase related protein, Hamajima et al,1996, Gene 180: 157-163), C-22 (Quach et al, 1997, Mol Brain Res. 46:329-332) or ULIP (Unc-33-Like protein, Byk et al, 1998, Eur J Biochem254: 14-24, and International Application WO 98/37 192), areintracellular signalling molecules that have, until now, been recognisedas being specific to the nervous system. These proteins have, inparticular, been described as being involved in controlling neuronaldevelopment and axonal growth.

Five proteins of this family have been identified to date: CRMP1, CRMP2,CRMP3, CRMP4 and CRMP5.

Certain members of the family have been associated with humanneurodegenerative disorders. In Alzheimer's disease, high levels ofphosphorylated CRMP2s are thus observed, in conjunction with neurofibrilplaques (Yoshida et al, 1998, J Biol Chem 273: 9761-9768).

In paraneoplastic neurological syndromes (PNSs), which areneurodegenerative disorders, patients develop auto-antibodies (anti-CV2antibodies) that recognise CRMPs (Honnorat et al, 1999, Eur J Neurosci11: 4226-4232).

Consequently, it has been proposed to use these proteins in thediagnosis and treatment of cancers and PNSs (FR 2 805 540 and WO 98/37192).

Yu et al, 2001 (Annals of Neurology, 49(2): 146-154), report,furthermore, the presence of anti-CRMP5 neuronal auto-antibodies incases of lung cancer and thymoma.

Unexpectedly, the inventors have revealed the presence of CRMPs in Tlymphocytes. More particularly, they have revealed CRMPs 1, 2 and 4 athigh levels in the T lymphocytes in patients affected by dysimmunepathologies. In such cases, it appears that the presence of theseproteins is increased when it is associated with a fatal anomaly or anabnormal proliferation of lymphocytes.

Furthermore, the inventors have also observed a markedly increasednuclear translocation of CRMP2 in lymphocytes infected with HTLV-1,probably rendering them hyperproliferative, or T lymphocytes of patientshaving an immune deficiency related to the Fas/Fas ligand system. Thistranslocation would correspond to a highly phosphorylated form of CRMP2,recognised more particularly by its specific antibody.

Finally, as will emerge from the following examples, the presence ofthese proteins has also been characterised in thymic epithelial cells,from the embryonic stage. This discovery of CRMPs in immature epithelialcells is one of the rare examples of intracellular signalling protein inthe thymus.

Furthermore, the disappearance of CRMPs in the adult thymus and theirinduction in the T cells after stimulation of the TCR indicates arelationship to the “rearrangement” of the T cell receptor (TCR) and theeducation of the thymocytes.

Consequently, all of this new data attests to the intervention of CRMPsin a signalling route that leads to the proliferation, death, maturationand education of lymphocytes, and therefore attests to their involvementin regulating the immune response.

These observations are all the more unexpected given that, until now,CRMPs had only been considered, in particular as far as treatment, isconcerned, as potential targets for treating pathologies of the centralnervous system (CNS).

It is clear that this discovery opens up new perspectives in terms oftreatment. It will now be conceivable to act on the proliferation or thedeath of T cells and/or to act on the cells of the thymus andautoimmunity by intervening at the level of the CRMPs in lymphocytes.

In such cases, the present invention relates specifically to the use ofCRMPS as targets for the treatment, prognosis and/or diagnosis ofpathologies related to a dysfunction of the immune system and, inparticular, the proliferation of T cells.

It relates, in particular, to modulating the expression or the activityof one or more CRMPs in such a way as to modulate the apoptosis,proliferation or migration of the cells.

An alteration of the CRMPs, such as, for example, a modification of thephosphorylation of CRMP2, is thus likely to result in ahyperproliferation or a cell death.

Similarly, stimulation of CRMPs seems to be an effective means forrestoring deficient signalling and improving lymphocyte survival, as inthe case of HIV infection.

Finally, controlling the hyperexpression of CRMPs seems to be crucialfor reducing the proliferation of lymphocytes that have been rendered“hyper”-proliferative, as in the case of an HTLV-1 infection or a Fasmutation. This inhibition might also improve the autoimmune processesthat involve T lymphocytes that recognise self determinants(autoreactives), and the attacks on the CNS related to the invasion ofthe CNS by these T lymphocytes (TSP/HAM related to HTLV-1, encephalitisrelated to measles, multiple sclerosis).

In certain cases, it is therefore desired to increase or stimulate theexpression or the activity of CRMPs (for example, in the case oflymphocytes infected with the AIDS HIV virus). In other cases, on theother hand, it is desired to suppress or inhibit the expression or theactivity of CRMPs, in order, for example, to reduce the proliferation orthe migration of lymphocytes, for example in the case of a lymphoma.

It may also be advantageous to block CRMP activity, in order to blockthe effect of the pathological prion protein (PrP).

In a first embodiment, the invention relates to the use of at least oneprotein of the CRMP family, a polypeptide fragment or biologicallyactive derivative thereof, or of a nucleotide sequence or sequencefragment that encodes said protein, for producing a pharmaceuticalcomposition intended for treating pathologies related to a dysfunctionof the immune system.

In another embodiment, the invention relates to the use of an antisensesequence that is capable of hybridising specifically with a sequencethat encodes in the manner of a nucleotide manner for said protein or anantibody directed against said protein, for producing a pharmaceuticalcomposition intended to treat pathologies related to a dysfunction ofthe immune system.

In another embodiment, the invention relates to the use of a peptidesequence that is capable of acting with said protein, or pharmaceuticalcompounds that are capable of controlling the expression of saidprotein, and/or their partners and/or their interactions, for producinga pharmaceutical composition intended to treat pathologies related to adysfunction of the immune system.

The invention also concerns a method for preventing and/or treating apathology related to a dysfunction of the immune system, this methodcomprising the administration of a therapeutically effective quantity ofan agent that modulates the expression or the activity of a CRMP, inconjunction with a pharmaceutically acceptable vehicle, to a patientrequiring a treatment of this type.

Details of these embodiments are given below.

Targeted Pathologies

According to the present invention, the pathologies that are targeted,from the point of view of treatment or diagnosis, are related to adysfunction of the immune system and, in particular, to a dysfunction ofthe proliferation of the cells of the immune system, in particular Tcells.

More precisely, these pathologies comprise:

-   -   T leukaemias (in particular adult T leukaemia) and lymphomas        (i.e. the malignant proliferation of T cells);    -   viral infections, such as an infection with herpes virus,        measles virus, Epstein-Barr virus, HTLV-1 (i.e.        neuroinflammatory diseases associated with infection with the        HTLV1 retrovirus, tropical spastic paraparesis or myelopathy        associated with HTLV-1-TSP/HAM), or an infection with the HIV        virus (AIDS virus, also called neuro-AIDS). More generally, any        viral infection that may result in an invasion of the CNS by T        lymphocytes (encephalitis related to measles, etc.) is targeted.    -   prion diseases.

Dysimmune diseases related to Fas and FasL mutation andneuroinflammatory diseases associated with an activation of lymphocytesare also targeted.

It is also conceivable to treat autoimmune diseases (and, in particular,those related to Fas) by acting, via CRMPs and their various biologicalpartners, on the maturation of the T lymphocytes over the thymus.

Examples of these autoimmune pathologies include, in particular,rheumatoid arthritis, myasthenia gravis, lupus erythematosus, asthma,multiple sclerosis, or any immune disorder involving an immunerecognition and the target of cells themselves or of tissues, andresulting in one or more inflammatory responses. Multiple sclerosis isthe most common of the autoimmune diseases.

More generally, the invention relates to the treatment or the diagnosisof demyelinating neuroinflammatory diseases.

According to a preferred embodiment of the invention, the patient is ahuman, preferably an adult, but the treatment method of the inventionmay also be applied to mammals or other vertebrates.

Modulation of CRMPs

According to the present invention, it is desired to modulate theactivity or the expression of CRMPs. This modulation may be direct orindirect.

A direct modulation of the CRMP activity is a modulation that is carriedout by direct action on the activity and/or the expression of theprotein itself. Agents that are capable of directly modulating CRMPactivity are either agonists or antagonists, and may also be referred toas “direct activators” or “direct inhibitors”, respectively.

The term “agonist” therefore refers to an agent that increases theactivity, while an “antagonist” denotes an agent that inhibits theactivity of the protein.

According to a particular embodiment, such agonists or antagonists arecapable of modulating the interaction of the CRMP with endogenousmolecules, which usually act directly upstream or downstream of the C ina signalling cascade. This may, for example, be a CRMP-semaphorin,CRMP-plexin, CRMP-kinase or alternatively CRMP-(protein of thecytoskeleton) interaction.

Agents of this type are, for example, antibodies directed against theCRMP or aptamers.

An alteration of the interaction between two homologous or heterologousCRMPs is another example of modulation of CRMP activity. An interactionbetween “homologous” proteins denotes an interaction between at leasttwo identical types of CRMPs, such as, for example, CRMP2-CRMP2homodimers.

Interaction between “heterologous” CRMPs denotes an interaction betweenat least two different CRMPS, for example CRMP2-CRMP5 heterodimers.

Agents that are capable of directly modulating the expression of a CRMPinclude agents that alter (i.e. increase or decrease) the level ofproduction of the CRMP. These agents may, for example, be a CRMPpolypeptide or a nucleic acid sequence encoding this protein, or agentscapable of modulating the transfection and/or the translation of CRMPgenes, such as antisense nucleic acid sequences or inhibitingdouble-stranded RNA.

An indirect modulation of CRMP activity is a modulation that is carriedout by acting on the activity and the expression of extracellular orintracellular endogenous agents, which usually act upstream (inducer) ordownstream (effector) of the CRMP, as a signalling cascade. An inducerof a CRMP is, for example, a semaphorin, in particular semaphorin 3A(Sema 3A) or semaphorin 4D (Sema 4D). Examples of effectors includetyrosine kinase, GTPases of the Rho or Rac family, and transferases(transglutaminase).

Other proteins that are capable of interacting with CRMPS, which may beidentified in pathological samples, such as cerebrospinal liquid orbrain biopsies, in a patient (human or animal) affected by animmunological disorder, also form part of the invention. Agents thatallow indirect modulation of the activity or the expression of a CRMP tobe obtained may easily be selected by a person skilled in the art, forexample in the light of the types of direct modulator described above.

According to the present invention, and unless otherwise stated, theterms “agent” or “test compound” may refer to one or more structurallydefined molecules, such as polypeptides, oligonucleotides or aninorganic or organic molecule of an endogenous or exogenous kind. Theseagents may also be undefined compounds, such as cell or tissue extracts,or biological liquids of animal or vegetable origin.

The use of CRMPs or nucleic acid proteins encoding a CRMP is beneficialwhen seeking to increase the quantity of at least one of the CRMPs. Itis also conceivable to use a compound or a mixture of compounds, ofsynthetic or natural origin, that activates or inhibits the expressionor the action of these CRMPs.

Examples of this type of compound include, in particular, molecules thatare capable of acting as a biological partner of CRMPs, using, forexample, CRMPs as an intracellular signalling molecule.

CRMPs

The CRMPs considered in particular, according to the invention, are theproteins CRMP1, CRMP2, CRMP3, CRMP4 and CRMP5.

The present invention preferably relates to the use of at least one CRMPselected from the amino acid sequences of the human proteins CRMP1,CRMP2, CRMP3, CRMP4 and CRMP5, which are shown in FIG. 8.

According to the present invention:

-   -   the protein CRMP1 refers, in particular, to a protein comprising        the amino acid sequence shown in FIG. 8, and also all of the        polypeptide fragments or corresponding derivatives.    -   the protein CRMP2 refers, in particular, to a protein comprising        the amino acid sequence shown in FIG. 8, and also all of the        polypeptide fragments or corresponding derivatives.    -   the protein CRMP3 refers, in particular, to a protein comprising        the amino acid sequence shown in FIG. 8, and also all of the        polypeptide fragments or corresponding derivatives.    -   the protein CRMP4 refers, in particular, to a protein comprising        the amino acid sequence shown in FIG. 8, and also all of the        polypeptide fragments or corresponding derivatives.    -   the protein CRMP5 refers, in particular, to a protein comprising        the amino acid sequence shown in FIG. 8, and also all of the        polypeptide fragments or corresponding derivatives.

The derived polypeptides refer to any polypeptide variant of the aboveproteins, or any other molecule resulting from a genetic modificationand/or of a chemical nature, of one of the previously specifiedsequences, i.e. that is obtained by mutation, deletion, addition,substitution and/or any chemical modification of a single amino acid orof a limited number of amino acids, as well any isoform sequence, saidderived, modified or isomorphic sequence having retained at least one ofthe properties rendering it biologically active.

Also included are homologous sequences, defined as:

i) sequences at least 70%, preferably 80%, more preferably 90%, similarto human CRMP sequences (as shown in FIG. 8);

ii) sequences encoded by a homologous nucleic acid sequence, i.e. anucleic acid sequence hybridising with a sequence encoding human CRMPsor its complementary sequence, under stringent hybridisation conditions.

The term “similar” refers to the perfect likeness or identity betweenthe compared amino acids, but also to the imperfect likeness that isdescribed as similarity. This study of similarities in a polypeptidesequence takes into account conservative substitutions, which aresubstitutions of amino acids of the same category, such as substitutionsof amino acids with non-charged side-chains (such as asparagine,glutamine, serine, threonine and tyrosine), of amino acids with basicside-chains (such as lysine, arginine and histidine), of amino acidswith acid side-chains (such as aspartic acid and glutamic acid), and ofamino acids with apolar side-chains (such as glycine, alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan andcysteine).

More generally, the term “homologous sequence of amino acids” refers,therefore, to any sequence of amino acids that differs from the humansequence of FIG. 8, by substitution, deletion and/or insertion of anamino acid or a small number of amino acids, in particular bysubstitution of natural amino acids with unnatural amino acids or pseudoaminoacids, in positions such that these modifications do notsignificantly impair the biological activity of the CRMP.

Homology is generally determined using a sequence-analysis softwarepackage (for example, Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705). Similar amino acid sequencesare aligned, in order to obtain the maximum degree of homology (i.e.identity or similarity, as defined above). For this purpose, it may benecessary to introduce gaps into the sequence artificially. Once theoptimal alignment has been produced, the degree of homology isestablished by recording all of the positions for which the amino acidsof the two compared sequences are identical, in relation to the totalnumber of positions.

The “biological activity” of CRMPs includes any biological property ofthese proteins. The activity may, for example, be determined byevaluating axonal growth inhibition in response to semaphorins, inparticular Sema 3A or Sema 4D, and/or by evaluating inhibition of thegrowth of oligodendrocytes and/or the migration of cells of the immunesystem in response to semaphorins. Also included are the immunologicalproperties of CRMPs, in particular the capacity to cause the productionof antibodies, of the anti-CV2 type of antibodies, in PNSs. Alsoincluded is CRMP enzyme activity, direct or indirect, or as a substrateof another enzyme.

Also concerned are the various forms of proteins that are consideredcapable of being recognised by their respective antibodies. These may betheir dimeric, phosphorylated and/or truncated form.

In FIG. 5, the presence of a particular form of phosphorylated CRMP2 isthus demonstrated by a Western blot, using a specific antibody, in thehyperproliferative T lymphocytes infected with the HTLV1 (C91PL)retrovirus, compared to the control lymphocytes (Jurkat).

The proteins CRMP2 and CRMP5 are particularly preferred as therapeutictargets in the present invention. The protein CRMP5, in particular,seems to be an especially beneficial target for the diagnosis and/ortreatment of pathologies of the prion disease type.

CRMPs, individually or in a mixture, may be associated with apharmaceutically acceptable vehicle within a pharmaceutical composition,which may be used for preventing or treating a dysfunction of the immunesystem.

Nucleic Acids

The invention also covers the use of any sequence of isolated nucleicacids encoding one of the proteins CRMP1, CRMP2, CRMP3, CRMP4 and CRMP5,or nucleotide fragments or sequences deriving from one of the sequences,owing to the degeneration of the genetic code or owing to mutation,deletion or insertion of at least one nucleotide, said derived sequenceshaving a biological activity that is practically identical to that ofthe protein in question.

The various nucleotide or peptide sequences of the invention may be ofartificial or non-artificial origin. They may be DNA or RNA sequences,obtained by screening sequence banks by means of probes developed on thebasis of the original sequences. Banks of this type may be prepared byconventional techniques of molecular biology known to a person skilledin the art.

The nucleotide sequences according to the invention may also be preparedby chemical synthesis or alternatively by mixed methods, including thechemical or enzymatic modification of sequences obtained by screeningbands. These nucleotide sequences permit the production of nucleotideprobes that are capable of hybridising strongly and specifically with asequence of nucleic acids, of a genomic DNA or a messenger RNA encodinga peptide according to the invention, or a biologically active fragmentthereof.

Also included are homologous sequences, defined as:

i) sequences similar to at least 70%, preferably 80%, more preferably90%, of a sequence encoding a human CRMP (as shown in FIG. 8), or

ii) sequences hybridising with a sequence encoding a human CRMP or itscomplementary sequence, in strict hybridisation conditions.

Preferably, an homologous nucleotide sequence of this type hybridisesspecifically with complementary sequences of the sequence encoding aCRMP of FIG. 8, under stringent conditions. The parameters defining thestringent conditions depend on the temperature at which 50% of thepaired strands separate (Tm).

For sequences comprising more than 30 bases, Tm is defined by therelationship: Tm=81.5+0.41 (% G+C)+16.6 Log (concentration ofcations)−0.63 (% formamide)−(600/number of bases) (Sambrook et al,1989). For sequences less than 30 bases in length, Tm is defined by therelationship: Tm=4 (G+C)+2 (A+T).

Under appropriate conditions of stringency, with which the non-specificsequences do not hybridise, the hybridisation temperature may preferablybe from 5 to 10° C. below Tm, and the hybridisation buffers used arepreferably solutions with high ionic strength, such as a 6×SSC solution,for example.

The term “similar sequences”, used above, refers to the perfect likenessor identity between the compared nucleotides, but also to the imperfectlikeness that is described as similarity. This study of similarities innucleic sequences distinguishes, for example, between purines andpyrimidines.

Homologous sequences of this type include the sequences of genes ofmammals other than humans, encoding a CRMP, preferably of a primate, abovine, ovine or porcine, or alternatively of a rodent, as well as theallelic variants.

A nucleic acid encoding a CRMP may, in particular, be used in genetherapy, in conjunction with a pharmaceutically acceptable vehicle,within a pharmaceutical composition.

Antisenses

The use of anti-CRMP1, CRMP2, CRMP3, CRMP4 and/or CRMP5 antisensesequences is, for its part, favoured when it is desired to block theexpression of the CRMP in question.

The nucleotide sequences encoding the proteins CRMP1, CRMP2, CRMP3,CRMP4 or CRMP5 may be used for producing antisense sequences that arecapable of hybridising specifically with a sequence of nucleic acidsincluding messenger RNA, which may be used in gene therapy.

The invention therefore also covers the use of antisense sequences thatare capable of at least partially inhibiting the production of the CRMPsidentified above

These sequences may be formulated in pharmaceutical compositions thatmay be used in gene therapy.

Anti-CRMP Antibodies

The invention also relates to the use of monoclonal or polyclonalantibodies, or fragments thereof, chimeric or immunoconjugatedantibodies obtained from a CRMP selected from the proteins CRMP1, CRMP2,CRMP3, CRMP4 and CRMP5, and biologically active polypeptide derivativesor fragments of CRMPs.

The antibodies may be used for blocking the expression of the CRMP inquestion.

The polycolonal antibodies may be obtained according to the protocoldescribed in the examples. These antibodies may, however, also beobtained using conventional methods.

The polycolonal antibodies may be obtained from the serum of an animalimmunised against a polypeptide according to the common operatingprocedures.

According to one embodiment of the invention, an appropriate peptidefragment, as defined above, which is capable of being coupled via areactive residue to a protein or another peptide, may be used as anantigen. Rabbits were immunised with the equivalent of 1 mg of peptideantigen, according to the procedure described by Benoit et al., PNASUSA, 79, 917-921 (1982). The animals were treated with injections of 200μg of antigen at four week intervals and bled 10 to 14 days later. Afterthe third injection, the antiserum was examined, in order to determineits capacity to bind to the antigen peptide radioactively-labelled withiodine, prepared according to the chloramine-T method, and then purifiedby chromatography over a carboxymethyl cellulose (CMC) ion exchangecolumn. The antibody molecules were then collected from the mammals andisolated to the desired concentration by methods that are well-known toa person skilled in the art, for example using DEAE Sephadex in order toobtain the IgG fraction.

In order to increase the specificity of the polyclonal serum, theantibodies may be purified by immuno-affinity chromatography, usingpolypeptides that immunise in the solid phase. The antibody is placed incontact with the polypeptide that immunises in the solid phase for asufficient period of time to make the polypeptide immuno-react with theantibody molecule, in order to form an immunological complex in thesolid phase.

Examples of the sequences used to produce these antibodies include, inparticular, those shown in FIG. 8.

These are:

-   -   peptide 6 (CMRP1): SEQ ID no 22;    -   peptide 3 (CMRP2): SEQ ID no 23;    -   peptide 4 (CMRP2): SEQ ID no 24;    -   peptide 7 (CRMP4): SEQ ID no 25;    -   peptide 8 (CRMP3): SEQ ID no 26;    -   peptide 5 (CRMP5): SEQ ID no 27;    -   peptide 9 (CRMP2 C-terminal): SEQ ID no 28.

Anti-peptide 4 is more specific to CRMP2, which is particularlyadvantageous given the strong homology between CRMPs, which makesspecific antibodies difficult to produce. The peptide comprises a serinethat is a potential phosphorylation site and that is likely tofacilitate detection of the phosphorylated form.

Anti-peptide 9 (CRMP2 C-terminal) is also specific to one part of theprotein CRMP2, which is truncated in certain pathological circumstances.

Anti-peptide 5 is specific to CRMP5 and therefore advantageously allowsCRMP5 to be distinguished from other CRMPs.

Monoclonal antibodies, which may, for example, be obtained by theconventional hybridoma culture method of Köhler and Milstein, Nature,256, 495-497, (1975), may also be used.

The antibodies may be chimeric antibodies, humanised antibodies, or Faband F(ab′)2 fragments. They may also be in the form of immunoconjugatesor labelled antibodies.

Anti-CRMP antibodies may be used in conjunction with a pharmaceuticallyacceptable vehicle, in pharmaceutical compositions that may be used forthe prevention or treatment of a dysfunction of the immune system. Theymay also be used as a diagnostic tool.

Interaction with Other Partners

It is also conceivable to intervene in the signalling cascade by using acompound or mixture of compounds, of synthetic or natural origin, thatis capable of inhibiting the action of said CRMPs and, moreparticularly, of blocking interaction, either between two of saidproteins, or between one of said proteins and its natural biologicalpartners.

More generally, it is conceivable to use CRMPs as baits for identifyingnew therapeutic targets in biological extracts, of the lipid or biopsytype, from patients affected by autoimmune or neuroinflammatorydiseases, lymphomas or T leukaemias.

The invention also relates to the use of at least one CRMP as adiagnostic tool for detecting, identifying and/or analysing at least onebiological partner of a CRMP in biological extracts from patientsaffected by autoimmune or neuroinflammatory diseases, lymphomas or Tleukaemias.

The inventors have thus demonstrated that CRMPs, which are intracellularsignalling molecules, react with the PrP prion protein, which isinvolved in membrane signalling.

It is likely that the modulation of the expression of CRMPs in thelymphocytes via the PrP protein would occur, in the degenerationprocess, at the level of the transfer of pathogenic information from theimmune system toward the central nervous system. This aspect of theinvention is illustrated by example 4 (below).

As a biological partner of CRNPs, the PrP protein may also be abeneficial target for modulating the activity of CRMPs. The therapeuticapproach in question would therefore consist in blocking interactionbetween the PrP and the CRMP, in particular CRMP5.

In such cases, another aspect of the invention relates to apharmaceutical composition comprising, specifically by way of an activeingredient, a compound or mixture of compounds, of synthetic or naturalorigin, that is capable of targeting at least one CRMP and/or ofinhibiting the natural interaction of at least one CRMP and/or a CRMPdimer with the PrP prion protein.

According to one particular embodiment of the invention, the CRMP inquestion is CRMP5.

In the sense of the invention, the compound or mixture of compoundsacts, more particularly, by blocking interaction between the twoproteins in question. The so-called aptamer compounds are particularlysuitable for this purpose. These are molecules that have the ability tobind to other molecules with great affinity and specificity. Examples ofthis type of compound include, in particular, peptide aptamers.

Pathologies that are capable of being treated according to this variantare sporadic, acquired or genetic prion diseases or, more generally, anydisease involving PrP in cellular signalling.

Compounds possessing a stimulating or inhibiting activity with regard toCRMPs may be selected using a screening method, in which the compound tobe tested is placed in contact with a CRMP, and the interaction betweenthe two proteins determined.

The invention also relates to a method for the in vitro screening ofmolecules that may be used for the treatment of prion diseases, whereina molecule to be tested is placed in contact with a PrP prion proteinand a CRMP, and the capacity of the molecule to inhibit the interactionof the CRMP, or of a CRMP dimmer, with the PrP protein is evaluated, aninhibiting action on this interaction being indicative of a moleculethat may be used for the treatment of prion diseases.

Administration

The optimal modes of administration, dosages and galenical forms of thepharmaceutical compositions according to the invention may be determinedaccording to the criteria that are generally taken into account whenestablishing a suitable therapeutic treatment for a patient, such as,for example, the age and body weight of the patient, his general stateof health, treatment tolerance, recorded side effects, etc.

Preferably, the pharmaceutical compositions according to the inventionmay be administered systemically, preferably intravenously,intramuscularly, intradermally or orally.

Generally, a therapeutically or prophylactically effective quantityvarying from around 0.1 μg to around 1 mg of CRMP may be administered tohuman adults.

The invention also relates to a pharmaceutical composition comprising anucleic acid, as defined above, encoding a CRMP or an antisense nucleicacid and a pharmaceutically acceptable vehicle, said composition beingintended to be used in gene therapy. The nucleic acid, which ispreferably inserted into a, generally viral, vector (such asadenoviruses and retroviruses), may be administered in naked form,without any vehicle promoting the transfer to the target cell, such asanionic liposomes, cationic lipids, microparticles, for example goldmicroparticles, precipitating agents, for example calcium phosphate, orany other agent facilitating transfection. In this case, thepolynucleotide may simply be diluted in a physiologically acceptablesolution, such as a sterile solution or a sterile buffer solution, inthe presence or in the absence of a vehicle.

Alternatively, a nucleic acid of the invention may be associated withagents that facilitate transfection. It may, inter alia, be (i)associated with a chemical agent that modifies cellular permeability,such as bupivacaine; (ii) encapsulated in liposomes, possibly in thepresence of additional substances facilitating transfection; or (iii)associated with cationic lipids or silica, gold or tungstenmicroparticles.

If the nucleic acid constructions of the invention cover microparticles,said microparticles may be injected intradermally or intraepidermally,by the “gene gun” technique (WO 94/24263).

The quantity to be used as medicine depends, in particular, on thenucleic acid construction itself, on the individual to whom this nucleicacid is administered, on the mode of administration and type offormulation, and on the pathology. Generally, a therapeutically orprophylactically effective quantity varying from around 0.1 μg to around1 mg, preferably from around 1 μg to around 800 μg, preferentially fromaround 25 μg to around 250 μg, may be administered to human adults.

The nucleic acid constructions of the invention may be administered byany conventional administration route, such as, in particular,parenterally. The choice of the route of administration depends, inparticular, on the selected formulation.

Diagnostic Methods

The invention also relates to an in vitro method for the prognosisand/or diagnosis of a pathology related to a dysfunction of the immunesystem, the method comprising the revelation, in cells of the immunesystem, i.e., in particular, lymphocytes, dendritic cells and monocytestaken from a patient (for example, from the blood or the brain), of anabnormal expression or location of the CRMP relative to the controllymphocytes.

The invention relates, more precisely, to a method for the prognosisand/or diagnosis of autoimmune diseases, lymphomas, adult leukaemia,neuroinflammatory diseases, which may or may not be related to a viralinfection, and/or prion diseases, characterised in that the presence ofat least one CRMP, the expression, sequence or location of which ismodified relative to the control lymphocytes obtained from healthysubjects, is revealed in lymphocytes taken from an individual.

Any pathology related to a dysfunction of T lymphocytes, as describedabove, may thus be diagnosed.

In particular, it may be determined whether an individual is a carrierof, or is likely to develop, a pathology selected from:

-   -   T leukaemias and (T) lymphomas;    -   viral infections, such as herpes virus, measles virus,        Epstein-Barr virus, HTLV-1 or HIV;    -   prion diseases;    -   and demyelinating neuroinflammatory diseases.

The expression of CRMPs may be evaluated by various techniques that arewell-known to a person skilled in the art. The RNA encoding the proteinsmay be detected and quantified using specific nucleotide probes.

According to the first embodiment, the invention relates to a method forthe in vitro diagnosis of a pathology related to a dysfunction of theimmune system, or of a predisposition to develop a pathology related toa dysfunction of the immune system, comprising the stages consisting in:

-   -   bringing together a biological sample containing mRNA, obtained        from lymphocytes in a patient, with specific oligonucleotides        allowing the amplification of all or part of the transcript of        the encoding gene for a CRMP;    -   amplifying said transcript,    -   detecting and quantifying the amplification products;        a modification of the transcript rate of the CRMP relative to a        normal control being indicative of a pathology related to a        dysfunction of the immune system, or of a predisposition to        develop such a pathology.

The products of the encoding genes for the CRMPs may also be analysedusing corresponding antibodies, as described above, and located in thecell.

According to this second embodiment, the invention relates to a methodfor the in vitro diagnosis of a pathology related to a dysfunction ofthe immune system, or of a predisposition to develop a pathology relatedto a dysfunction of the immune system, for detecting or measuring therate of CRMP expression and/or activity in a sample of lymphocytes of apatient, using anti-CRMP antibodies. This method comprises the placingin contact of at least one antibody directed against CRMP with saidsample under conditions which allow the possible formation of specificimmunological complexes between the CRMP and said antibody or antibodiesand the detection of the specific immunological complexes possiblyformed and/or the inhibition of CRMP activity by the antibody.

Alternatively, the presence of the anti-CRMP antibodies may bedetermined by means of the CRMPs or their epitopic fragments which maybe labelled in such a way that the complexes formed between the proteinsand said antibodies may then be detected easily in biological samples.

The following examples and figures are presented to illustrate but notlimit the present invention.

FIGURES

FIG. 1: comparison by RT-PCR analysis of the level of expression of themRNA encoding CRMP-4, -2 and -1 in mononucleated or control lymphocytesand cells or in those derived from patients affected by dysimmunepathologies. The level of expression of the CRMP mRNA is standardisedrelative to the level of expression of the GAPDH ubiquitary gene. Theresults are expressed as a relative value as a ratio of pixels betweenthe amplicon of each CRMP and the amplicon of G3PDH.

FIG. 2: electrophoretic profile of the CRMP 1, 2 and 4 proteins in aJurkat lymphocyte line compared with a Dev nerve cell line.

FIG. 3: immunocytochemical detection of the subcellular location of theCRMP2 protein in mononucleated blood cells (PBL) of control patients orpatients infected with HTLV-1 or having an immune deficiency associatedwith the Fas/Fas ligand system, or infected with HIV.

FIG. 4: characterisation of the nuclear presence recognised by the antiCRMP-2 (peptide 4) in the hyperproliferative T lymphocytes.

FIG. 4 a: examination after immunodetection of CRMP-2 andcounter-staining by the intercalator of the Dapi DNA in the Jurkat Tcells (controls) and the C 8166 cells (T infected by HTLV-1 which do notproduce viruses).

FIG. 4 b: examination of the immunodetection of CRMP-2 by confocalmicroscopy in the CEM cells (control line T) and the C91PL cells (lineinfected with HTLV-1).

FIG. 5: demonstration by a Western blot using specific anti-CRMP2antibodies (peptide 4) of the presence of a particular form ofphosphorylated CRMP2 in the hyperproliferative T lymphocytes (C91PL)compared with the control lymphocytes (Jurkat).

FIG. 6: immunohystochemical analysis of the expression of the CRMP5 andCRMP2 proteins over the human foetal thymus (A, B, C) and in a thymoma(D, E, F).

FIG. 7: characterisation by two-hybrid tests by conjugation of theinteraction between the bovine PrP protein and the human CRMP5 protein.

FIG. 8: amino acid sequences of the human proteins CRMP1, CRMP2, CRMP3,CRMP4 and CRMP5. This figure also shows the sequences of the peptidesselected to produce the antibodies specifically directed against theCRMPs.

FIG. 9 shows the migration kinetics of T lymphocytes transfected or nottransfected with a vector encoding CRMP-2.

FIG. 10 reports the number of T cells which have migrated in threeexperiments, after over-expression of CRMP-2 gfp.

FIG. 11 reports the number of T cells which have migrated in twoexperiments, after transfection of CRMP-2 plasmids associated with cmycand mutated CRMP-2 (Delta 381CRMP2-cmyc).

EQUIPMENT AND METHODS 1. Cells and Cell Culture

-   -   SI cells: the T lymphocytes are either lymphocytes established        in lines. (IL-2 independent) or lymphocytes in primary culture,        the growth of which necessitates the presence of IL-2. These        lymphocytes are cultivated (37° C., 5% CO₂) in suspension in a        supplemented saline medium, namely in SVF (10%) in the case of        lymphocyte lines, in AB type human serum (SAB 10%) and in IL-2        in the case of primary cultures of T lymphocytes. Peripheral        blood lymphocytes (PBL) have also been freshly isolated from the        blood of control patients or subjects, separated over a Ficoll        gradient and recovered after a phase of adhesion of the        monocytes/macrophages.    -   Treatments of the T lymphocytes: for certain experiments, the        freshly isolated PBLs were treated with agonistic CD3 antibodies        (10 μg/ml) which mimic the activation of the lymphocytes by an        antigen via the T receptor (TCR) or by the phytohemagglutinin        (PHA-10 μg/ml) (C. Malcus).

2. Examination of Transcripts (mRNA) by the RT-PCR (ReverseTranscription and Polymerase Chain Reaction) Method

-   -   Isolation of the total RNA: this experiment is carried out        systematically at 4° C. to avoid degradation of the RNAs by the        Rnases. RNA was extracted from cell cultures or from freshly        isolated lymphocytes to which a solution of RNAzol is added (2        ml per 10⁶ cells). The RNAzol contains the phenol and the        guanidium isothiocyanate and lyses the cells. The phase is        separated using chloroform (400 μl per 2 ml of RNAzol). After        homogenisation (vortex) then 5 minutes of rest, the sample is        centrifuged at rest (5 minutes), then centrifuged at 12,000 rpm,        for 30 minutes at 4° C. The RNAs present in the aqueous phase        (measured volume) are precipitated first by addition of an        identical volume of isopropanol (15 min at 4° C.) then        centrifugation (12,000 rpm for 15 minutes at 4° C.). A second        precipitation is carried out by adding a volume of 0.3 M sodium        acetate pH 5.2 and three volumes of absolute ethanol at −20° C.,        to the pellet. The precipitate is collected by centrifugation        (12,000 rpm, 15 minutes, 4° C.). the salts are removed by adding        800 μl of 80% ethanol at −20° C., to the pellet followed by        centrifugation. After removing all traces of ethanol, the pellet        is dissolved in 100 μl of distilled water and stored at −80° C.        The RNAs are analysed by measuring the optical density using a        spectrophotometer at 260 and 280 nm (1 unit of OD at 260 nm=40        μg of RNA). The ratio of the ODs obtained at 260 nm and at 280        nm should be close to 2, which is an indication of good RNA        extraction without a trace of proteins.    -   Reverse transcription: reverse transcription (RT) is the stage        preliminary to iterative polymerisation which uses a polymerase        DNA that is incapable of polymerising DNA from RNA. RT is        carried out using a reverse transcriptase, the Rtase of the MuLV        virus, and is initiated by a primer which allows the elongation        and synthesis of one complementary strand of DNA of the RNA by        the enzyme. In a comparison study of the products of        amplification of various mRNAs, the use of the non-specific        primers (oligodT) was found to be more suitable, affording the        possibility of leading the various specific PCRs from a same        sample of RT.    -   500 ng of total RNA diluted in 7 μl of water (QS) are incubated        for 10 minutes at 70° C. to allow the denaturation of the        secondary structures, then the tubes are immediately plunged        into ice to prevent renaturation. To these 70 μl of total RNA        there are then added 1 μl of oligodT (100 mg/μl) and 12 μl of        “incubation” medium containing 0.5 mM of dNTP, 4 μl of RT        buffer, 40 U of RNAsine (Rnase inhibitor), 10 mM of        dithiothreitol or DTT (denaturant), 1 μl of MuLV-Rtase (200        U/μl). The reaction mixture is incubated for 90 minutes at        42° C. The products of RT are then diluted to 10/10^(th) in        distilled water and stored at −20° C.    -   The polymerisation chain reaction (PCR): iterative        polymerisation is a technique which involves repeating the        polymerisation cycles of a segment of a DNA which is of        interest. The first stage of each cycle is a denaturation stage        carried out at 95° C. The second stage is a hybridisation or        attachment stage with specific primers of the segment which        enclose the region to be amplified at a temperature at which 50%        of the DNA are in double-stranded form and the remaining 50% in        single-stranded form (Tm). The third stage is the stage of        elongation at 72° C. which is the optimum temperature for the        activity of thermostable DNA dependent DNA polymerase, Taq        polymerase. The parameters which have to be taken into        consideration as a priority to optimise the PCR are the choice        of the primers (performed by the Primer 3 software and validated        by comparison with indexed human sequences, Blast), the        hybridisation temperature or Tm, the concentration of MgCl₂ and        the number of cycles. All these conditions were perfected before        carrying out the PCRs and are summarised in the following Table:

Primers for RT-PCR Number Probe for the Southern blot Tm [MgCl₂] ofcycles CRMP-4 Sense: Antisense: Probe: 62° C. 2 mM 28gcaagtgtaggaaggcacgct ggcagctctggaacgtgaaga cctcagccttgttcttcacg SEQ IDNO 1 SEQ ID NO 2 SEQ ID NO 3 CRMP-2 Sense: Antisense: Probe: 62° C. 3 mM22 tcacatcagaactcctgtgg catgagtggaggaactttc ccatcaccacccttgtctct SEQ IDNO 4 SEQ ID NO 5 SEQ ID NO 6 CRMP-1 Sense: Antisense: Probe: 62° C. 3 mM26 tcatgctgaatccacctcgg cctctgaggcagttgacgg gacatcgccaaggactgact SEQ IDNO 7 SEQ ID NO 8 SEQ ID NO 9 CRMP-3 Sense: Antisense: Probe: 62° C. 3 mM30 gccgcccctaccagagacc gtgcagcgacagccagat cggagaaaacctcatcgt SEQ ID NO10 SEQ ID NO 11 SEQ ID NO 12 CRMP-5 Sense: Antisense: Probe: 62° C. 3 mM28 ctgggagagaggagtggttg gaagtctccLccctggacct gttttgtggccgttaccagt SEQ IDNO 13 SEQ ID NO 14 SEQ ID NO 15 Actine Sense: Antisense: Probe: 62° C. 3mM 25 ggactlcgagcaagagatgg acatctgctggaaggtggac aagtactccgtgtgtggatcggSEQ ID NO 16 SEQ ID NO 17 SEQ ID NO 18 G3PDH Sense: Antisense: Probe:62° C. 3 mM 23 ggctctccngmacatcatcc ggagattcaglgtggtgggacatcnngaaggtggtgaagcag SEQ ID NO 17 SEQ ID NO 18 SEQ ID NO 19

-   -   Each sample of PCR contains 10 μl of RT diluted to 1/10^(th) and        40 μl of Mix, 5 μl of PCR 1× buffer, 2 or 3 μl of MgCl₂, 4×1 μl        of dNTP, 1 μl of each primer (sense and antisense), 0.4 μl of        Taq polymerase, QS 50 μl of distilled water. The PCR comprises a        first stage of denaturation for: 5 min at 95° C. then a        predetermined number of cycles for each pair of primers (cf.        Table) (1 min at 95° C., 1 min 15 at 62° C., 1 min 30 at 72°        C.). The PCR ends with an elongation stage of 15 min at 72° C.        and the PCR products are stored at −20° C.    -   Visualisation of the RT-PCRs: Southern blot: it is carried after        separation of the amplification products by electrophoresis and        then transfer to a membrane and hybridisation with an internal        radioactively labelled probe. The migration of the various PCR        products is effected by depositing 10 μl of each sample on a        1.5% agarose gel in a TBE (Tris-Borate-EDTA) buffer 0.5×        containing ethidium bromide (0.5 μg/ml at the end) and        application of 100 V. Once migration has occurred, the cDNA are        transferred onto a nylon membrane in TBE 0.5× buffer by a        semi-dry electrotransfer method. After transfer (15V—45 min),        the DNA is fixed to the membrane by a solution of NaOH (0.4N—2        min) then neutralised (SSC 6×—10 min). The internal probe, which        is specific for the DNA fragment amplified by PCR (cf. Table) is        radioactively labelled at 5′ by a terminal kinase (T4 kinase)        with γ32P-ATP. 2 μl are incubated for 10 min at 37° C. with 5 μl        of forward buffer, 1 μl of T4 kinase, 2 μl of γ32P-ATP and 15 μl        of distilled water. Kinase reaction is stopped at 4° C., the        radioactively labelled probe is then purified over an exclusion        column (Bio-Rad Laboratories) which retains the free        nucleotides. The transfer membranes are prehybridised for 30 min        at 42° C. by incubation in the hybridisation medium (SSC 6×,        Denhardt 2×, 25 mM phosphate buffer, 25 mM disodium ethylene        diamine tetraacetate or EDTA, SDS 0.1%, 250 μg/ml DNA of salmon        sperm) in order to saturate all the non-specific sites.        Hybridisation is carried out for 30 min at 42° C. with the        internal probe. The membranes are then washed at 42° C. in media        of increasing stringency: 10 min in SSC6×/0.1% SDS, 20 min in        SSC 2×/SDS 0.1% and 10 min in SSC 0.5×/0.1% SDS. The        radioactively labelled membranes sealed in a elastic sheet are        then placed in an autoradiography cassette equipped with an        amplifying screen. The radioactively labelled bands of cDNA are        visualised by reading using a Phosphorimager and are quantified        using computer software (Quant Image).

3. Immunodetection of the CRMPs

-   -   The anti-peptide antibodies: some specific anti-peptide        polyclonal antibodies of the 5 CRMPs were prepared after        selection of an immunogen situated in a non-homologous zone for        the other CRMPs and injection in a rabbit. The specificity of        each serum for a given CRMP was checked by the Western blot        method on the recombinant CRMPs. The sera of each of these        rabbits, taken before immunisation thereof (pre-immune sera),        are negative in the Western blot in the recombinant proteins        obtained in Ecolitranes formed or transfected Hela cells and are        used as a control. The optimum dilutions for the use thereof        were previously determined by the Western blot method or by        immunocytochemistry.    -   Western blot: this technique involves the electrophoretic        separation of the proteins then transfer onto a membrane which        allows a protein which is of interest to be visualised after the        fixing of a specific antibody revealed by a secondary antibody        coupled to the peroxidase. Western blots are produced on protein        extracts from lysates of immune or nerve cells in culture or        freshly isolated. The cells are generally lysed in a buffer        without detergent (20 mM Tris-HCl, 10% sucrose, 1 mM EDTA, 5 mM        EGTA) and a cocktail of anti-proteases (Complete™ 1×) and        sometimes in RIPA buffer (10 mM Tris-HCl pH 7.2, 150 mM of NaCl,        1% of Triton 100×, 0.1% of SDS, 1 mM of EDTA, 1% of sodium        deoxycholate, Complete 1×). Homogenisation of the cell lysates        was then completed by sonication at 80 Hz. After protein        determination, a solution of 1 or 2 μg/μl of proteins is        preserved at −20° C. The sample of deposit (final 40 μg of        proteins) is denatured by the addition of a reducing buffer        (0.0625 M Tris-Hcl pH 6.8, 1% SDS, 10% glycerol, 0.1 M        dithiothreitol DTT, bromophenol blue) and heating (5 min at 95°        C.). The proteins are separated over SDS acrylamide gel        (10%-0.1% respectively). After migration under 100 V, the        proteins are electrotransferred onto a nitrocellulose membrane        by discontinuous transfer using 3 buffers: buffer 1, 0.3 M Tris        base, 20% methanol); buffer 2 (25 mM Tris base, 20% methanol);        buffer 3 (25 mM Tris base, 40 mM EACA, 20% methanol) allowing        better transfer of all the proteins, whatever their molecular        weight. The membranes are incubated for 5 min in a solution of        Ponceau red and trichloroacetic acid which allows the proteins        to be fixed and visualised. The membranes are then saturated for        1 hour at ambient temperature in a solution of phosphate buffer        (PBS)—0.1% Tween 20 and 5% skimmed milk. Immunodetection of the        CRMPs with the anti-CRMP polyclonal rabbit sera diluted in PBS,        0.1% Tween 20 and 1% milk (CRMP-4: 1/100; CRMP-2: 1/500; CRMP-1:        1/500; CRMP-3: 1/500; CRMP-5: 1/200) or pre-immune rabbit sera (        1/200) is effected at 4° C. for one night. The blots are then        rinsed (3×5 min) in PBS, 0.1% Tween 20 and 1% milk then        incubated for 1 hour with a specific secondary antibody of the        rabbit IgG and coupled with peroxicase ( 1/50000^(th)−1        hour-ambient temperature). After 3 rinses (5 min) in PBS, 0.1%        Tween 20 and 1% milk, the blot is revealed in a black chamber        using an electrochemiluminescence kit (Covalab) and after        printing on photographic film.    -   During an experiment, the samples were treated with a        phosphatase for 1 hour at 37° C. before being separated on an        acrylamide/SDS gel: 2 μl of CIP (calf intestinal phosphatase at        20 units/μl) alkaline phosphatase are added to 20 μl of protein        sample and to 2 μl of buffer, allowing enzyme activity.    -   Immunocytochemistry (ICC): the CRMPs were detected on a cellular        scale by immunocytochemistry. The lymphocytes, which are        non-adhesive cells, are spread by centrifugation (cytospin-600        rpm-5 min) whereas the Dev nerve cells are cultivated on        Permanox slides (Labteck). Fixing of the cells by acetone (−20°        C., 5 min) has the object of fixing the proteins and making the        cells permeable to the entry of the reactive antibodies. These        conditions enabled the CRMP 1, 2, 3, 4 and 5 to be detected. The        non-specific sites are blocked with a 0.1% solution of BSA (30        minutes at ambient temperature). First contact of the cells        fixed on slides is made with the primary antibody (anti-CRMP        produced in rabbits, diluted in PBS, pH 7.4) (1 h-37° C. in a        moist chamber). After washing three times in phosphate buffer        (PBS, pH 7.4, 5 min), the specific secondary antibody of the        rabbit IgG is placed in contact (1 h-ambient temperature) and        washed three times in PBS for 5 min. The counter-staining of the        nuclei is then effected by incubation in a Dapi solution        (nucleic intercalator-0.025 μg/ml-1 min). The slides are mounted        in glycerol buffered to pH 7.4. The results of these analyses        are shown in FIG. 2 to 8.

EXAMPLE 1 Evaluation of the Expression of CRMP 1, 2 and 4 inMononucleated Lymphocytes and Cells, Control Lymphocytes and Cells orLymphocytes and Cells Derived from Patients Affected by DysimmunePathologies

-   -   mRNA (relative to GAPDH)

A first evaluation was made by titrating the corresponding mRNA. Theresults are presented in detail in FIG. 1 and summarised in Table Ibelow.

Comparative Profile, Considering all the Samples for the Same CRMP:

TABLE I CRMP2 CRMP4 CRMP1 Control T lines + 0 0 T/HTLV-1 lymphocytes +++++ +++ “Control” patients ± 0 ± lympho. Apopt. defic. patients ± 0 +/++lympho. HIV patients ++ ++ ++

The foregoing analysis is completed by detecting CRMP 1, 2 and 4proteins in T lymphocytes and nerve cells by a Western blot on a celllysate (Dev: nerve cells; Jurkat; T lymphocyte line) in non-detergentbuffer and using the specific antipeptide polyclonal antibodies of eachCRMP.

The results are presented in FIG. 2.

It will be noted that the anti-CRMP-1, -2 and -4 detect a plurality ofisoforms shared by the T lymphocytes and the nerve cells.

EXAMPLE 2

Immunocytochemical detection of the subcellular location of the CRMP2protein in mononucleated blood cells (PBL) of control patients orpatients infected with HTLV-1 or having an immune deficiency associatedwith the Fas/Fas ligand system or infected with HIV.

The anti CRMP-2 antibody was previously prepared and isolated by theprotocol described in item 3 of the preamble of “equipment and method”.

The results are illustrated in FIG. 3.

It will be noted that:

-   -   the location of CRMP-2 is essentially cytoplasmic in the        lymphocytes (PBL) isolated from a control subject or from a        subject infected with HIV-1; it may also be nuclear in the        lymphocytes isolated from patients infected with HTLV-1 or in        the PBL of a dysimmune patient (Fas deficiency);    -   CRMP-2 is expressed more in the lymphocytes of patients than in        those of the control subject cultivated in the IL2 and therefore        preactivated.

By examination after immunodetection of CRMP-2 and counter-staining bythe intercalator of Dapi DNA, it is noted that the expression is largelynuclear in the hyperproliferative T lymphocytes which are chronicallyinfected with RTLV-1. Examination of CRMP-2 immunodetection by confocalmicroscopy reveals a very weak nuclear presence in the control Tlymphocytes and a majority nuclear presence in the T lymphocyteschronically infected with HTLV-1 (FIG. 4).

To conclude, the expression of CRMP2 is increased in the nuclei ofproliferative lymphocytes. On the other hand, CRMP2 remains cycloplasmicin patients infected with HIV. The location is nuclear in some cells indysimmune patients (Fas deficient). FIGS. 3 and 4 show the resultsobtained.

Similarly, the nuclear presence of a protein recognised by this sameantibody has been checked in the hyperproliferative T lymphocytes by aWestern blot on cell fractions enriched in nuclei.

EXAMPLE 3 Characterisation by Immunohystochemistry of the Expression ofthe CRMP5 and CRMP2 Proteins Over the Thymus

The proteins were characterised by means of their respective antibodies.

The analysed cells are cells of normal human fetus thymus and in athymoma in an adult patient.

As a control, the same immunohystochemistry was carried out on humanthymus of a six-week old embryo. The thymus is deep-frozen and sectionedin a cryostat (15 μm). The sections are fixed for 15 minutes in acetone.Immunohystochemistry is carried out under the same conditions as on thebrain.

This analysis reveals an expression of CRMP2 and CRMP5 in the thymicepithelial cells which is normal at the embryonic stage. The expressiondisappears in normal adults and may be induced in the case of tumoralpathology (thymoma).

EXAMPLE 4 Characterization of the Interaction Between Bovine PrP Proteinand Human CRMP5 Protein

The interaction between bovine PrP protein and human CRMP5 protein wascharacterised by two-hybrid tests by conjugation.

The cDNA which are encoding these two proteins (complete or differentderived forms) were cloned in a “bait” vector and a “prey” vector. Thebait vector pEG202 (Gyuris et al, 1993, Cell 75, 791-803) comprises thegene encoding the marker HIS3, an origin of replication 2μ and directsthe expression of proteins merged with the range of association with theDNA of the LexA protein (1-202), under the control of the constituentpromoter ADHp. The prey vector, pJG 4-5 (U.S. Pat. No. 5,580,736),comprises the gene encoding the marker TRP1, an origin of replication 2microns and directs the expression of merged proteins to a nuclearlocation sequence (NLS) to the transcription activating range (B42) andto the epitope HA under the control of the inducible promoter GALP. Forconvenience, the fusion NLS-B42-HA is designated “Act” here.

The strain EGY42 (MATα) (Golemis et al, 1992, Mol. Cell. Biol. 12,3006-3014) was co-transformed with the plasma vector pSH-18-34 (U.S.Pat. No. 5,695,941) comprising the marker gene URA3 and eight LexAoperators placed upstream of the reporter gene lacZγ and various baitvectors directing the expression of LexA, LexA-PrP, LexA-PrPm (mutationson PrP still to be determined), LexA-PrPtr (truncation of PrP still tobe determined), LexA-MAX (negative control) and LexA-CRMP5.

The strain EGY48 (MATα) was transformed with various prey vectorsdirecting the expression of Act, Act-PrP, Act-BaxΔTM (negative control)and Act-CRMP5.

These various strains were conjugated so as to create an interactionmatrix.

This matrix was replicated on an indicator medium (Ura⁻, His⁻, Trp⁻,galactose/X-gal) allowing expression of the baits and the preys,selection of the diploid exconjugants and revelation of the interactionphenotypes (colour blue, revealing β-galactosidase activity observedwhen the reporter gene lacZ is transcribed).

FIG. 7 shows the results obtained.

The following constructions were tested:

PrP: bovine PrP protein (AA 22-237),

PrP: bovine PrP protein (AA 22-237) containing various mutations whichare still to be determined,

PrPtr: bovine PrP protein (AA-22-?), truncated toward the middle of thesequence (the position of the codon is still to be determined),

MAX: negative interaction control

CRMP 5: complete human CRMP 5 protein

BAXΔTM: negative interaction control.

The interaction between the PrP proteins (various constructions detailedbelow) and the CRMP5 protein is detected when the PrP proteins areexpressed as baits and the CRMP5 protein is expressed as prey.LexA-CRMP5 and Act-Prp do not give an interaction phenotype. Thenegative controls (LexA-MAX and Act-BaxΔTM as well as LexA and Act) donot give an interaction phenotype with the PrP and CRMPs. Thehomodimerisation of CRMP5 provides the positive interaction control ofthis matrix.

EXAMPLE 5 Effect of the CRMP on the Migration of the T Lymphocytes

Method

The migration of the T lymphocytes (Jurkat) is evaluated aftertransfection or non-transfection of plasmid encoding various forms ofCRMP-2: gfb associated CRMP2: cmyc associated CRMP-2 and mutated CRMP-2,Delta381CRMP-2 (deletion of amino acids 381 to 572 containing on aTyrKin site, a SH3 binding site and the site of adhesion to the tubulinheterodimers (Fukata et al. Nat Cell Biol, 2002, Aug. 4(8):583-91). Thetransfection is verified by the detection of GFP and of cmyc in thecells before and after migration by a Western blot.

Migration takes place in a Boyden chamber (3 micron pores) for 18 hoursin the standard RPMI medium+foetal calf serum. 400,000 cells aredeposited in the upper well and the number of cells which have migratedinto the lower well is counted (triple wells, three experiments withCRMP-2gfp and two with cmyc CRMP2). The base level of migration is thatobtained with the cells transfected with a plasmid not containing theCRMP-2 insert.

The CRMP/vimentin association is visualised by immunoprecipitation and aWestern blot (IP vimentin Wester CRMP) and co-immunodetection onmigrating or non-migrating T lymphocyte cells (confocal microscopy).

Results

The over-expression of gfp CRMP-2 or cmyc CRMP-2 in the Jurkatsincreases the number of cells which have migrated in 6 hours and 18hours (FIGS. 9 and 10).

The mutation of Delta381 CRMP-2 (site containing TyrKin and the tubulinebinding site) amplifies the migration, suggesting a role of this rangein migration (FIG. 11).

The CRMP-2 is the partner of vimentin (cytoskeleton protein), suggestingthe participation of CRMP-2 in the reclassification of the cytoskeleton.

1. A method for in vitro screening for a pathology involving a dysfunction of the T lymphocytes, said method comprising: a) evaluating the level of expression, the location, or the phosphorylation of a Collapsin Response Mediator Protein (CRMP) in cells of the immune system taken from a patient; and b) comparing the level of expression, location or the phosphorylation of said CRMP with the level of expression, location or the phosphorylation of said Collapsin Response Mediator Proteins in control cells of the immune system from a healthy subject, wherein an increased expression, or modified location, or modified phosphorylation of said CRMP in the cells of the immune system taken from said patient as compared to the expression, the location or the phosphorylation of said CRMP in the control cells of the immune system obtained from said healthy subject is indicative of a pathology involving a dysfunction of the T lymphocytes, wherein the Collapsin Response Mediator Protein is CRMP2 or CRMP5, and wherein said pathology is selected from the group consisting of: T leukaemias and lymphomas, viral infections, wherein the viral infection is selected from the group consisting of measles virus and HTLV-1; and multiple sclerosis.
 2. The method according to claim 1, wherein said cells of the immune system are selected from lymphocytes, dendritic cells and monocytes.
 3. The method according to claim 1, further comprising the steps of: contacting a biological sample containing mRNA, obtained from lymphocytes in a patient, with oligonucleotides allowing the amplification of all or part of the transcript of the gene encoding a CRMP; amplifying said transcript; and detecting and quantifying the amplification products, wherein an increase of the transcript rate of the CRMP relative to a normal control is indicative of said pathology involving a dysfunction of the T lymphocytes, or of a predisposition to develop such a pathology.
 4. The method according to claim 1, wherein the level of expression, the location or the phosphorylation of the CRMP in lymphocytes is evaluated by contacting of at least one antibody directed against the CRMP with a sample of lymphocytes under conditions which allow the possible formation of specific immunological complexes between the CRMP and said antibody or antibodies and the detection of the specific immunological complexes possibly formed and/or the inhibition of CRMP activity by the antibody, wherein an increased expression or modified location or modified phosphorylation of said CRMP in the lymphocytes taken from said patient as compared to the expression, the location or the phosphorylation of said CRMP in the lymphocytes obtained from said healthy subject is indicative of said pathology involving a dysfunction of the T lymphocytes.
 5. The method according to claim 1, wherein the increased expression or modified location or modified phosphorylation of the CRMP is detected with an antibody directed against the CRMP.
 6. The method according to claim 5, wherein the antibody directed against the CRMP is an antibody directed against a peptide of sequence SEQ ID NO:24, SEQ ID NO:28 or SEQ ID NO:27.
 7. The method according to claim 1, wherein an increased expression of said CRMP in the cells of the immune system taken from said patient as compared to the expression of said CRMP in the control cells of the immune system obtained from said healthy subject is indicative of said pathology involving a dysfunction of the T lymphocytes.
 8. The method according to claim 1, wherein an increased nuclear translocation of said CRMP in the cells of the immune system taken from said patient as compared to the location of said CRMP in the control cells of the immune system obtained from said healthy subject is indicative of said pathology involving a dysfunction of the T lymphocytes.
 9. The method according to claim 1, wherein an increased phosphorylation of said CRMP in the cells of the immune system taken from said patient as compared to the phosphorylation of said CRMP in the control cells of the immune system obtained from said healthy subject is indicative of said pathology involving a dysfunction of the T lymphocytes. 